Proteins of the wnt signaling pathway and uses thereof in the diagnostic and treatment of hypopigmentation disorders

ABSTRACT

The present invention relates to the identification of proteins of the WNT signaling pathway as therapeutic targets of pigmentation disorder and as biomarkers of pigmentation status. The invention in particular relates to products and methods for treating a hypopigmentation disorder. The invention also relates to products and methods for detecting, diagnosing, staging or monitoring the course of hypopigmentation disorder and is particularly suited for human subjects.

The present invention relates to the identification of protein of theWNT signaling pathway as therapeutic targets of pigmentation disorders,typically of hypopigmentation disorders, and as biomarkers ofpigmentation status. It further relates to corresponding diagnostic andtherapeutic applications as well as disease's management applications.The invention in particular relates to products and methods for treatinga pigmentation disorder, typically a hypopigmentation disorder such asvitiligo. The invention also relates to products and methods fordetecting, diagnosing, staging or monitoring the course of pigmentationdisorder, typically a hypopigmentation disorder such as vitiligo, and isparticularly suited for human subjects. The invention also relates tobinding reagents specific for proteins of the WNT signaling pathway,compositions and devices containing the same, as well as to their usesfor pigmentation disorder detection, diagnostic, staging, monitoring,imaging or treatment, as well as for drug development. The presentinvention more specifically relates to the assessment of a pigmentationdisorder, typically a hypopigmentation disorder, using a DNA or a mRNAencoding a protein of the WNT signaling pathway, or the protein itself,as a biomarker.

BACKGROUND

Pigmentation disorders are disturbances of human skin color, either lossor reduction (depigmentation or hypopigmentation) which may be relatedto loss of melanocytes or to the inability of melanocytes to producemelanin or transport melanosomes correctly, or increase(hyperpigmentation) which is caused by an excessive production ofmelanin by melanocytes. Melanocytes are located at the lower layer (thestratum basale) of the skin's epidermis, the middle layer of the eye(the uvea), the inner ear, meninges, bones, and heart.

Vitiligo is an acquired depigmentation of the skin inducing a markedalteration of the quality of life of affected individuals. This diseaseis characterized by destruction of melanocytes that occurs mainly in theskin and results in the appearance of well circumscribed white macules.There are two types of vitiligo, i.e., segmental vitiligo locatedunilaterally on an area of the face, upper body, legs or arms, which ingeneral does not change; and generalized vitiligo, which has more orless often bilateral symmetrical spots on areas of repeated friction orpressure and may become increasingly important over the years. The exactphysiopathological mechanism that leads to the destruction ofmelanocytes is still elusive, and involves autoimmunity (Passeron T,Ortonne J P 2005; Spritz 2007).

Vitiligo is common and affects 1% to 2% of the general population. Formany patients with vitiligo, the disfigurement caused by the disease hasa great impact on their quality of life (Ongenae K et al. 2006).

Halting the disease progression and repigmenting the lesional skinrepresent the two faces of the therapeutic challenge in vitiligo. Sofar, none of them has been successfully addressed. Oxidative stress andimmune system in genetically predisposed individuals participate to thecomplex pathophysiology of vitiligo.

Currently, there are several therapeutic modalities that can be proposedfor the treatment of vitiligo.

Treatments such as narrow-band UVB (Nb-UVB), excimer light, topicalsteroids, topical tacrolimus or pimecrolimus and combination approaches(with phototherapy and topical steroids or calcineurin inhibitors) canprovide cosmetically acceptable repigmentation (>75%) [Lepe, 2003;Ostovari, 2004; Passeron, 2004; Taieb, 2013]. Unfortunately,repigmentation, consisting in vitiligo skin in the differentiation andproliferation of new melanocytes, remains difficult to achieve in mostcases. Some localizations, such as hands and feet, are almost impossibleto fully repigment with the current approaches. In addition, it is stillvery difficult to compare the efficacy of different treatment modalitiesand the results of different studies on the same treatment because: (i)most published studies are uncontrolled; and (ii) there is not agenerally accepted biometric tool to assess disease severity andresponse to treatment. Recently, animal models using reactive T-cellsagainst melanocyte antigens provided interesting data on the immunereaction potentially involved in the depigmentation of vitiligo skin butthis model is not adapted to study mechanisms of melanocytesdifferentiation and repigmentation in vitiligo skin [Mosenson, 2013][Rashighi, 2014].

In the light of limited therapies and the lack of information about thephysiopathology of hypopigmentation disorders such as vitiligo, there isa clear need for identifying new pharmacological markers allowing theircorrect and early diagnostic as well as their adequate management, andfor new therapeutic targets allowing their prevention, attenuation ortreatment.

BRIEF DESCRIPTION OF THE INVENTION

Surprisingly, inventors herein demonstrate for the first time thatactivating or stimulating the WNT pathway allows the repigmentation ofdepigmented skin lesions, in particular of vitiligo lesions.

A first object of the invention thus relates to an activator of at leastone protein of the WNT signaling pathway for use for treating ahypopigmentation disorder such as vitiligo in a subject, typically forrepigmenting a hypopigmentation lesion. Herein described is also the useof an activator (agonist) of at least one protein of the WNT signalingpathway for the preparation of a composition for treatinghypopigmentation disorder such as vitiligo.

The present invention also relates to the assessment or monitoring ofpigmentation status, typically hypopigmentation disorder such asvitiligo, using at least one, for example two, three, four, five or six,protein(s) of the WNT signaling pathway as biomarker(s). The applicationshows that analysis or measurement of at least one protein of the WNTsignaling pathway allows the early detection or the follow-up ofpatients having pigmentation disorder with remarkably high relevancy,robustness, sensitivity and selectivity.

The invention is particularly suited to detect or monitor pigmentationdisorders in human subjects, in particular a hypopigmentation disordersuch as vitiligo (particularly the different stages of vitiligo).

An object of the invention relates to a method for the in vitro or exvivo detection, diagnosis or staging of a pigmentation disorder,typically of a hypopigmentation disorder such as vitiligo, in a subjectsuspected of suffering of a pigmentation disorder, comprising analysingthe expression of at least one protein of the WNT signaling pathway in abiological sample from the subject, said analysis providing informationon the presence or stage of a pigmentation disorder in the individual.

Another object of the invention relates to a method for the in vitro orex vivo detection or diagnosis of a hypopigmentation disorder such asvitiligo, comprising the following steps of:

a) analysing the expression of at least one protein of the WNT signalingpathway in a biological sample from a subject suspected of suffering ofa pigmentation disorder,

b) analysing the expression of the at least one protein of the WNTsignaling pathway in a biological sample from a healthy subject,

c) comparing the expressions of the at least one protein of the WNTsignaling pathway as analysed in steps a) and b),

an expression of the at least one protein of the WNT signaling pathwayin the biological sample from the subject suspected of suffering of ahypopigmentation disorder lower than the expression of the at least oneprotein of the WNT signaling pathway in the biological sample from thehealthy subject being an indicator of the presence of a hypopigmentationdisorder in the individual suspected of suffering of a hypopigmentationdisorder, thereby detecting or diagnosing the hypopigmentation disorder.

Another object of the invention relates to a method for monitoring invitro or ex vivo the course of a hypopigmentation disorder affecting anindividual, wherein the method comprises a step of comparing theexpression of at least one protein of the WNT signaling pathway in afirst biological sample taken from a subject at t0 to the expression ofthe at least one protein of the WNT signaling pathway in a secondbiological sample taken from said subject at t1, a decrease of theexpression of the at least one protein of the WNT signaling pathway inthe sample taken at t1 being an indicator of the progression of thehypopigmentation disorder in said subject and an increase of theexpression of the at least one protein of the WNT signaling pathway inthe sample taken at t1 being an indicator of the regression of thehypopigmentation disorder in said subject.

A further object of the invention relates to a method for monitoring invitro or ex vivo the efficacy of a drug or composition for treating ahypopigmentation disorder, comprising comparing the expression of atleast one protein of the WNT signaling pathway in a first biologicalsample from a subject identified as having one or more of the symptomsof a hypopigmentation disorder before any treatment of thehypopigmentation disorder to the expression of the at least one proteinof the WNT signaling pathway in a second biological sample of the samesubject who has been exposed to a drug or composition for treating ahypopigmentation disorder, an increase in the expression of the at leastone protein of the WNT signaling pathway in the second biological samplebeing an indicator of efficacy of the drug or composition for treatingthe hypopigmentation disorder, and a decrease or an absence ofmodulation in the expression of the at least one protein of the WNTsignaling pathway in the second biological sample being an indicator ofinefficacy of the drug or composition for treating the hypopigmentationdisorder.

Also herein described is an in vitro or ex vivo screening method of anactivator a protein of the WNT signaling pathway, comprising determiningthe ability of a drug candidate to increase (activate or stimulate), oron the contrary to decrease or suppress, the expression and/orbiological function of the protein of the WNT signaling pathway and ifthe ability is confirmed the identification of the drug candidate as anactivator (agonist), or on the contrary as an inhibitor (antagonist), ofthe protein of the WNT signaling pathway.

Another in vitro or ex vivo screening method of modulators of a proteinof the WNT signaling pathway, preferably activators, comprises thefollowing steps of:

a) contacting a biological sample exhibiting a hypopigmentation disorderlesion, a biological sample exhibiting the healthy condition, or amixture of said samples, with one or more drug candidates to be tested;

b) detecting the expression and/or biological function of a protein ofthe WNT signaling pathway in the biological samples or mixture ofsamples of step a) and comparing said expression or biological functionwith the expression or biological function of the protein of the WNTsignaling pathway in a sample which has not been contacted with the oneor more drug candidates;

c) selecting as modulators drug candidates which activate or stimulate(activators), or on the contrary which decrease or suppress(inhibitors), the expression and/or biological function of the proteinof the WNT signaling pathway as measured in the samples or mixturesobtained in the end of step a).

The invention also relates to kits or devices suitable for implementingthe above methods, for example a device comprising at least onecomplementary nucleic acid, antibody, fragment or derivative thereofthat binds a protein of the WNT signaling pathway or a nucleic acidcoding for such a protein immobilized on a support.

The invention also relates to compositions comprising an inhibitor of aprotein of the WNT signaling pathway as herein described and totherapeutic or diagnostic uses thereof.

DETAILED DESCRIPTION OF THE INVENTION

A challenge in the treatment of pigmentation disorders is the lack ofefficient molecules allowing efficient differentiation and proliferationof new melanocytes in depigmented area of the skin (including hands andfeet), typically in vitiligo lesions, of a subject.

Another challenge is the lack of early, pre-symptomatic detection asvisible symptoms of the disorders generally present at advanced stages.

The Wnt signaling pathways are a group of signal transduction pathwaysmade of proteins that pass signals from outside of a cell through cellsurface receptors to the inside of the cell. Three Wnt signalingpathways have been characterized: the canonical Wnt pathway, thenoncanonical planar cell polarity pathway, and the noncanonicalWnt/calcium pathway. All three Wnt signaling pathways are activated bythe binding of a Wnt-protein ligand to a Frizzled family receptor, whichpasses the biological signal to the protein Dishevelled inside the cell.The canonical Wnt pathway leads to regulation of gene transcription, thenoncanonical planar cell polarity pathway regulates the cytoskeletonthat is responsible for the shape of the cell, and the noncanonicalWnt/calcium pathway regulates calcium inside the cell. Wnt signalingpathways use either nearby cell-cell communication (paracrine) orsame-cell communication (autocrine). They are highly evolutionarilyconserved, which means they are similar across many species from fruitflies to human.

Wnt signaling was first identified for its role in carcinogenesis, buthas since been recognized for its function in embryonic development. Theembryonic processes it controls include body axis patterning, cell fatespecification, cell proliferation, and cell migration. These processesare necessary for proper formation of important tissues including bone,heart, and muscle.

Here, inventors describe new therapeutic targets for use for treating ahypopigmentation disorder such as vitiligo.

In the context of the present invention, proteins of interest of the WNTpathway are advantageously selected from LEF (Lymphoid enhancer-bindingfactor), TCF (DNA-binding transcription factor), MITF(Microphthalmia-associated transcription factor), DCT (Dopachrometautomerase or dopachrome delta-isomerase, tyrosine-related protein 2),PAX3 (transcription factor 3 of the paired box (PAX) family) and BRn2transcription factor.

These proteins can also be used as biomarkers as they allow specific,reliable and sensitive detection and staging of pigmentation disordersin particular in human subjects (also herein identified as humanindividuals or human patients).

For the purpose of the present invention, the tem “marker” or“biomarker” designates a biological marker associated with the absence,presence or stage of a particular pathological or physiological state.The biological markers are in particular proteins, mRNAs or DNA.

Unless otherwise specified, LEF designates the LEF gene, the LEF mRNA orthe LEF protein as well as any fragment thereof. Specific examples ofLEF protein according to the invention include full length LEF protein,in particular LEF1, and any fragment thereof.

Similarly, TCF designates the TCF gene, the TCF mRNA or the TCF proteinas well as any fragment thereof. Specific examples of TCF proteinaccording to the invention include full length LEF protein and anyfragment thereof. The same is can be said from MITF, DCT, PAX3 and BRn2transcription factor.

Inventors performed a transcriptome and proteomic analysis on lesional,perilesional and non-depigmented skin of vitiligo patients compared tomatched skin controls of healthy subjects. Their results show that theWNT/β-catenin pathway, implicated in melanocytes differentiation, isaltered in vitiligo skin. They demonstrated that the oxidative stressdecreases WNT expression/activation in keratinocytes and in melanocytes.They developed an ex vivo skin model that remains functional up to 15days and confirmed the decreased activation of the WNT pathway in humanskin subjected to oxidative stress thanks to said model. Finally, usingpharmacological agents that activate the WNT pathway and pharmacologicalagents that inhibit GSK3B, they treated the ex vivo depigmented skinsfrom vitiligo patients and successfully induced the differentiation ofresident stem cells into pre-melanocytes thereby allowing repigmentationof vitiligo lesions to occur, even in areas such as hands and feets.

The invention now provides novel therapeutic targets as well asbiomarkers, namely proteins of the WNT regulatory pathway, preferablyselected from LEF, TCF, MITF, DCT, PAX3, BRn2 and any combinationthereof, for clinical applications, typically pigmentation statusassessment, in particular hypopigmentation disorder patient detection,diagnosis, treatment, monitoring and management.

Herein described is an activator (agonist) of at least one protein ofthe WNT signaling pathway for use for treating a hypopigmentationdisorder, in particular vitiligo, in a subject. Such an activator istypically for use for repigmenting a hypopigmentation lesion, inparticular a vitiligo lesion, for example a vitiligo lesion located inareas such as hands and feets.

Also described is the use of an activator of at least one protein of theWNT signaling pathway for the preparation of a composition for treatingan hypopigmentation disorder such as vitiligo, even in areas such ashands and feets.

Such an activator (agonist) of at least one protein of the WNT signalingpathway is advantageously selected from SKL2001, SKL9001, lithiumchloride (LiCl), CHIR99021, SB216763, SB415286, and(2′Z,3′E)-6-bromoindirubin-3-oxime (BIO), preferably from LiCl andCHIR99021. A preferred activator is LiCl.

Also herein described is a pharmaceutical composition comprising atleast one activator of at least one protein of the WNT signaling pathwayas herein described. Such a composition may be formulated into asuitable dosage form using technology well known to those skilled in theart. The pharmaceutical composition can comprise a pharmaceuticallyacceptable excipient, vehicle or carrier such as those widely employedin the art of drug-manufacturing. For instance, the pharmaceuticallyacceptable carrier may include one or more of the following agents:solvents such as olive oil, olive oil refined, cottonseed oil, sesameoil, sunflower seed oil, peanut oil, wheat germ oil, soybean oil, jojobaoil, evening primrose oil, coconut oil, palm oil, sweet almond oil, aloeoil, apricot kernel oil, avocado oil, borage oil, hemp seed oil,macadamia nut oil, rose hip oil, pecan oil, hazelnut oil, sasanqua oil,rice bran oil, shea butter, corn oil, camellia oil, grape seed oil,canola oil, castor oil, and combinations thereof, preferably olive oilrefined, emulsifiers, suspending agents, decomposers, binding agents,excipients, stabilizing agents, chelating agents, diluents, gellingagents, thickening agent such as beeswax and/or petroleum jelly,preservatives, lubricants, absorption delaying agents, liposomes,antioxidants such as butylhydroxytoluene or butylhydroxyanisole, and thelike. Preferably, the pharmaceutical composition is formulated into atopical formulation that can be directly applied to the skin, forexample, a skin suffering from vitiligo. The topical formulationsuitable for the pharmaceutical composition may be an emulsion, a gel,an ointment, a cream, a patch, an embrocation, an aerosol, a spray, alotion, a serum, a paste, a foam, or a drop. In one embodiment of thisapplication, the pharmaceutical composition is formulated into anexternal preparation by admixing the extract according to thisapplication with a base such as those that are well known and commonlyused in the art.

In some embodiments, the dosage and the frequency of administration ofthe pharmaceutical composition according to the herein describedapplication may vary depending on the following factors: the severity ofthe disease to be treated, the weight, age, physical condition andresponse of the subject to be treated. In further or additionalembodiments, the amount of the active ingredient(s), typicallyactivator(s) of at least one protein of the WNT signaling pathway, inthe pharmaceutical composition is in the range of about 0.001 to about1000 mg/kg body weight/day, for example, about 0.01 to about 500, 300,or 100mg/kg body weight/day.

Within the context of the invention, the use of at least one protein ofthe WNT signaling pathway for pigmentation disorder detection,diagnosis, staging or management (typically monitoring of the course ofthe pigmentation disorder) includes, without limitation, the use of theprotein (in any form, soluble or not, full length or not), or of anycoding nucleic acids, as a biomarker. This includes, e.g., the use ofany reagent to detect or quantify (i) the protein or any variant ormutant thereof, such as splicing variants or polymorphisms, and/or (ii)any nucleic acid encoding said proteins, such as DNA or RNA, saidprotein and/or nucleic acid levels being correlated to the pigmentationdisorder. The term also includes any measure of the expression level ofthe cited protein, and a comparison of the measured level to a referenceor mean value. The measured amount or level or information provides anindication regarding the specified pigmentation disorder in the subject.

A specific object of the invention relates to a method for the in vitroor ex vivo detection, diagnosis or staging of a hypopigmentationdisorder in a subject suspected of suffering of a hypopigmentationdisorder, comprising analysing the expression of at least one protein ofthe WNT signaling pathway in a biological sample from the subject, saidanalysis providing information on the presence or stage of ahypopigmentation disorder in the subject.

Another object of the invention relates to a method for the in vitro orex vivo detection or diagnosis of a hypopigmentation disorder,comprising the following steps of:

a) analysing the expression of at least one protein of the WNT signalingpathway in a biological sample from a subject suspected of suffering ofa hypopigmentation disorder,

b) analysing the expression of the at least one protein of the WNTsignaling pathway in a biological sample from a healthy subject,

c) comparing the expressions of the at least one protein of the WNTsignaling pathway as analysed in steps a) and b),

an expression of the at least one protein of the WNT signaling pathwayin the biological sample from the subject suspected of suffering of apigmentation disorder lower than the expression of the at least oneprotein of the WNT signaling pathway in the biological sample from thehealthy subject being an indicator of the presence of a hypopigmentationdisorder in the subject suspected of suffering of a hypopigmentationdisorder, thereby detecting or diagnosing the pigmentation disorder.

In the context of the invention, the subject is an animal, typically amammal, preferably a human being whatever its age or sex. The biologicalsample is typically a tissue sample, preferably a skin sample, such as abiopsy, taken from a subject. The biopsy may vary in size and ispreferably from 1 to 6 mm in diameter.

According to one particular and preferred embodiment, the sample is askin sample taken by means of tape stripping, such as with D-Squames,according to the method described in Wong R et al., “Analysis of RNArecovery and gene expression in the epidermis using non-invasive tapestripping”; J Dermatol Sci. 2006 November; 44(2):81-92; or in Benson NR, et al., “An analysis of select pathogenic messages in lesional andnon-lesional psoriatic skin using non-invasive tape harvesting”. JInvest Dermatol. 2006 October; 126(10): 2234-41; or else in Wong R etal., “Use of RT-PCR and DNA microarrays to characterize RNA recovered bynon-invasive tape harvesting of normal and inflamed skin”. J InvestDermatol. 2004 July; 123(1):159-67. According to the principle of tapestripping, the product used comprises a flexible translucent polymersupport and an adhesive. The product is applied repeatedly to the skinof the patient, preferably until loss of adhesion. The sample obtainedrelates only to the content of the outermost layers of the epidermis.

The analysis typically comprises determining the presence, absence oramount of the at least one protein of the WNT signaling pathway, theabsence of at least one protein of the WNT signaling pathway or anexpression thereof higher than a reference amount being indicative ofthe presence or stage of a hypopigmentation disorder in the individual.

The expression analysis or detection can be performed by any suitablemethod, known to those skilled in the art, such as western blotting,IHC, mass spectrometry (Maldi-TOF and LC/MS analyses), radioimmunoassay(RIA), ELISA or any other method known to those skilled in the art orelse by assaying the mRNA according to the methods customarily known tothose skilled in the art. The techniques based on the hybridization ofmRNA with specific nucleotide probes are the most customary [in situhybridation, FISH, Northern blotting, RT-PCR (Reverse TranscriptasePolymerase Chain Reaction), quantitative RT-PCR (qRT-PCR), RNaseprotection].

A method for analysing a protein content obtained in particularaccording to the previously herein described sampling method isdescribed in Patent Application WO2009/068825 (Galderma R&D). Since thismethod is rapid, non-invasive and relatively inexpensive, it isparticularly preferred. Quantification is performed in the skin sampleobtained on the flexible and adhesive support in order to detect the atleast one protein of the WNT signaling pathway of which the presence,the absence or the variation in amount or in concentration compared witha standard value is associated with the presence, with the progressionor with the absence of the (potentially suspected) pigmentationdisorder.

In a particular embodiment, analysing at least one protein of the WNTsignaling pathway comprises contacting a biological sample, or analiquot thereof, with a specific binding reagent that binds the at leastone protein of the WNT signaling pathway or nucleic acid encoding saidprotein and determining the presence or amount of the at least oneprotein of the WNT signaling pathway or of the corresponding nucleicacid bound to said binding reagent.

Selective or specific binding indicates that binding to another moleculecan be discriminated from (e.g., occurs with higher affinity or aviditythan) specific binding to the target biomarker. Preferred reagents donot bind, under selective condition, to any other unrelated human bloodprotein but the reference protein. Binding of a reagent to a referencemolecule can be tested according to methods well known by the skilledperson.

The binding reagent is typically a specific ligand selected from acomplementary nucleic acid, an antibody, an aptamer, and a fragment orderivative thereof.

In a particular embodiment, the binding reagent is an antibody. Theantibody may be a polyclonal or a monoclonal antibody, most preferably amonoclonal. It may be of various classes (e.g., IgG, IgE, IgM, etc.).The antibody may be of various animal origin, or human or synthetic orrecombinant. Furthermore, the term antibody also includes fragments andderivatives thereof, in particular fragments and derivatives of saidmonoclonal or polyclonal antibodies having substantially the sameantigenic specificity. Antibody fragments include e.g., Fab, Fab′2,CDRs, etc. Derivatives include humanized antibodies, human antibodies,chimeric antibodies, poly-functional antibodies, Single Chain antibodies(ScFv), etc. These may be produced according to conventional methods,including immunization of an animal and collection of serum (polyclonal)or spleen cells (to produce hybridomas by fusion with appropriate celllines).

Methods of producing polyclonal antibodies from various species,including mice, rodents, primates, horses, pigs, rabbits, poultry, etc.are well known from the skilled person. Briefly, the antigen is combinedwith an adjuvant (e.g., Freud's adjuvant) and administered to an animal,typically by sub-cutaneous injection. Repeated injections may beperformed. Blood samples are collected and immunoglobulins or serum areseparated.

Methods of producing monoclonal antibodies from various species aslisted above may be found, for instance, in Harlow et al. (Antibodies: Alaboratory Manual, CSH Press, 1988) or in Kohler et al. (Nature 256(1975) 495), incorporated herein by reference. Briefly, these methodscomprise immunizing an animal with the antigen, followed by a recoveryof spleen cells which are then fused with immortalized cells, such asmyeloma cells. The resulting hybridomas produce the monoclonalantibodies and can be selected by limit dilutions to isolate individualclones. Antibodies may also be produced by selection of combinatoriallibraries of immunoglobulins, as disclosed for instance in Ward et al.(Nature 341 (1989) 544).

Recombinant antibodies, or fragments or derivatives thereof, may beproduced by methods known per se in the art, for example byrecombination in a host cell, transformed with one or more vectorsenabling the expression and/or secretion of the nucleotide sequencesencoding the heavy chain or the light chain of the antibody. The vectorgenerally contains a promoter, translation initiation and terminationsignals, and suitable transcriptional regulatory regions. It is stablymaintained in the host cell and may optionally possess specific signalsfor secretion of the translated protein. These different components areselected and optimized by one of skill in the art according to the hostcell used.

In a preferred embodiment, the antibody directed against a protein ofthe WNT signaling pathway, as well as the fragment or derivative thereofis an antibody, fragment or derivative thereof which binds the humanprotein of the WNT signaling pathway or a fragment thereof (such as apeptide or epitope).

Particular examples of such antibodies are monoclonal antibodies.

Antibodies may be found or generated against a protein of the WNTsignaling pathway and used in the present invention. It should be notedhowever that the use of antibodies that bind an epitope of a protein ofthe WNT signaling pathway wherein said binding is at least partiallydisplaced by the corresponding human protein of the WNT signalingpathway is particularly preferred as well as a fragment or derivative ofsuch an antibody having the same antigen specificity.

For use in the invention, the antibodies may be coupled to heterologousmoieties, such as labels, tags, linkers, etc., typically to a detectablemoiety.

The invention also relates to kits or devices suitable for implementingthe above methods.

A typical device comprises at least one specific reagent, typically atleast one complementary nucleic acid, antibody, fragment or derivativethereof, that binds a protein of the WNT signaling pathway or a nucleicacid encoding said protein, said specific reagent being immobilized on asupport. Preferably the support is a membrane, a slide, a microarray, achip or a microbead.

A particular kit comprises a device as herein described and at least onereagent to perform, detect or quantify an immune reaction, in particularan antibody-antigen complex.

A further object of the invention relates to a method for monitoring invitro or ex vivo the course of a hypopigmentation disorder affecting anindividual, wherein the method comprises a step of comparing theexpression of at least one protein of the WNT signaling pathway in afirst biological sample taken from a subject at t0 to the expression ofthe at least one protein of the WNT signaling pathway in a secondbiological sample taken from said subject at t1, t1 being posterior tot0, a decrease of the expression of the at least one protein of the WNTsignaling pathway in the sample taken at t1 being an indicator of theprogression of the hypopigmentation disorder in said individual and anincrease of the expression of the at least one protein of the WNTsignaling pathway in the sample taken at t1 being an indicator of theregression of the hypopigmentation disorder in said subject.

Another object of the invention relates to a method for monitoring invitro or ex vivo the efficacy of a drug or composition for treating ahypopigmentation disorder, comprising comparing the expression of atleast one protein of the WNT signaling pathway in a first biologicalsample from a subject identified as having one or more of the symptomsof a hypopigmentation disorder before any treatment of thehypopigmentation disorder to the expression of the at least one proteinof the WNT signaling pathway in a second biological sample of the samesubject who has been exposed to a drug or composition for treating apigmentation disorder, an increase in the expression of the at least oneprotein of the WNT signaling pathway in the second biological samplebeing an indicator of efficacy of the drug or composition for treatingthe hypopigmentation disorder, and a decrease or an absence ofmodulation in the expression of the at least one protein of the WNTsignaling pathway in the second biological sample being an indicator ofinefficacy of the drug or composition for treating the hypopigmentationdisorder.

Also herein described is an in vitro or ex vivo screening method of amodulator of a protein of the WNT signaling pathway, comprisingdetermining the ability of a drug candidate to activate or stimulate, oron the contrary to decrease or suppress, the expression and/orbiological function of the protein of the WNT signaling pathway, and, ifthe ability is confirmed, the identification of the drug candidate as aninhibitor (antagonist), or on the contrary as an activator (agonist), ofthe protein of the WNT signaling pathway.

Another in vitro or ex vivo screening method of modulators of a proteinof the WNT signaling pathway, preferably activators, comprises thefollowing steps of:

a) contacting a biological sample exhibiting a hypopigmentation disorderlesion, a biological sample exhibiting the healthy condition, or amixture of said samples, with one or more drug candidates to be tested;

b) detecting the expression and/or biological function of a protein ofthe WNT signaling pathway in the biological samples or mixture ofsamples of step a) and comparing said expression or biological functionwith the expression or biological function of the protein of the WNTsignaling pathway in a sample which has not been contacted with the oneor more drug candidates; and

c) selecting as modulators drug candidates which activate or stimulate(activators), or on the contrary which decrease or suppress(inhibitors), the expression and/or biological function of the proteinof the WNT signaling pathway as measured in the samples or mixturesobtained in the end of step a).

The identified modulator will influence the biological function of theprotein of the WNT signaling pathway or a biological process modulatedby this marker. For screening purposes, the biological samplesadvantageously consist of transfected cells containing reporter genesoperating under the control of a promoter (totally or partially)controlling the expression of the gene encoding the protein of the WNTsignalling pathway. Alternatively, the promoter may be, at least inpart, synthetically assembled and contain adequate responsive elements.The ability of a compound to modulate the function of the gene encodingthe protein of the WNT signaling pathway, is evaluated by analyzing theexpression of the reporter gene.

The transfected cells may further be engineered to express the proteinof the WNT signaling pathway. The reporter gene may encode an enzymethat with its corresponding substrate, provides coloured product(s) suchas CAT (chloramphenicol acetyltransferase), GAL (beta galactosidase), orGUS (beta glucuronidase). It might be either luciferase or GFP (GreenFluorescent Protein). Reporter gene protein dosage or its activity istypically assessed by colourimetric, fluorometric or chemoluminescencemethods.

The selected modulator, preferably inhibitor or antagonist, can be apolypeptide, a DNA, a RNA, or a PNA (“Peptide nucleic acid”, i.e. aDNA-like structure with a polypeptidic chain substituted by purine andpyrimidine bases). Advantageously, the modulator is administered to apatient in a sufficient quantity so as the measure a plasmaticconcentration which is from about 0.01 μg/ml to about 100 μg/ml,preferably from about 1 μg/ml to about 5 μg/ml.

Other characteristics and advantages of the invention are given in thefollowing experimental section (with reference to FIGS. 1-11), whichshould be regarded as illustrative and not limiting the scope of thepresent application.

FIGURES

FIG. 1. Transcriptomic analysis of vitiligo patients highlights thedisappearance of melanocytes and the involvement of WNT signalingpathway without hint of immune system activation.

(A) Histogram of differentially expressed genes showing that 95% ofupregulated transcripts in vitiligo lesional skin has less than 2-folddifference in gene expression when compared to healthy skin.

(B) Ingenuity based canonical pathways enriched in vitiligo lesionalskin.

(C) Cluster analysis of microarray data from healthy skin (HS), nonlesional (NLS), perilesional (PLS) and lesional skin (LS) from vitiligosubjects. All genes significantly modulated (|FC|≤1.5 P<0.05) wereincluded in this analysis.

FIG. 2. IPA based analysis of WNT and MITF pathway involvement invitiligo lesional skin.

A transcriptional network linking Wnt signaling and Melanogenesispathways. Lesional vitiligo skin is both characterized by adownregulated expression of key transducer of Wnt signaling pathway(E.g. LEF1), and by the upregulation of negative regulators of Wntsignaling pathway such as p53 and TLE4 (groucho family member) andZBTB33/Kaiso (involved in transcriptional repression of wnt targetgenes)

LEF1/bCatenin regulates melanogenesis through SOX10 transcriptionalinduction which in turn regulates the expression of EDNRB, PCSK2 andPLP1 genes. LEF1/bCatenin also directs the transcription of MITF whichgoverns melanogenesis at the transcriptional level. LEF1/bCatenin isalso linked to melanogenesis via the IRF4 encoding gene. Thistranscription factor whose function is still unclear in melanocyte hasbeen shown to cooperate with MITF.

FIG. 3. qRT-PCR validation of loss of melanocytic gene expression:vitiligo lesional skin shows complete loss of expression of genesinvolved in the terminal differentiation of melanocytes and residualexpression of key melanogenic transcription factors or adhesionmolecules encoding genes (A).

Gene expression was measured by qRT-PCR and calculated for groupanalysis, i.e. NLS (Non Lesional Skin) or PLS (Peri-Lesional Skin) or LS(Lesional Skin) group compared to HS group (Healthy Skin) using a T-TESTlinear model without pairing. Modulated genes were consideredsignificantly modulated if both the |fold change| was ≥1.8 and the FDRvalue was <0.05.

The y-axis displays fold change in expression relative to the pool ofhealthy skin. The data were normalized relative to HPRT, ACTB and GAPDH.The error bars display the standard error of three replicates. *=p<0.05;**=p<0.01; ***=p<0.001; ****=p<0.0001; *****=p<0.00001.

TLDA validation set encompassed both genes related to terminaldifferentiation markers (PMEL, MLANA, TYRP1, DCT), transcription factorsinvolved in melanogenesis (MITF, FOXD3, KIT, NANOG), and adhesionmolecules (PLXNC1, PCDH11X, CDH2 and L1CAM).

qRT-PCR validation of LEF1 mRNA (B) and CXCL10 mRNA (C): LEF1 isselectively decreased in lesional skin samples whereas CXCL10 mRNA isupregulated in non lesional and perilesional skin biopsies compared tohealthy skin.

Gene expression was measured by qRT-PCR and calculated for groupanalysis, i.e. NLS or PLS or LS group compared to HS group (HealthySkin) using a T-TEST linear model without pairing. Modulated genes wereconsidered significantly modulated if both the |fold change| was ≥1.8and the FDR value was <0.05. The y-axis displays fold change inexpression relative to the pool of healthy skin. The data werenormalized relative to HPRT, ACTB and GAPDH. The error bars display thestandard error of three replicates. *=p<0.05 **=p<0.005

FIG. 4: Oxidative stress decreases WNT pathway in the skin.

NHM (A) and NHK (B) and whole skin in ex vivo culture (C) werestimulated 24 hours with 25 to 100 μM of H₂O₂. After mRNA extraction, areverse transcription was performed and the relative gene expression ofLEF1 was analyzed with qPCR.

FIG. 5: Development of an ex vivo skin model to study vitiligo skin.

(A) Six mm skin biopsies were obtained from abdominoplasty surgery andwere disposed on a transwell chamber to allow a semi-liquid cultureenvironment. The skin was stimulated with 20 μM to 1 mM of forkolinadded in the culture medium every second days during 4 to 15 days. After4, 11 and 15 days, the morphology of the skin was visualized bymicroscopy with a Hematoxylin—Eosin staining (X20).

(B) The ability of the skin to respond to melanogenic stimuli wasassessed using forskolin treatment.

The mRNA was extracted from biopsies and analyzed by RT-qPCR to quantifythe expression of melanogenic genes: MITF, DCT and tyrosinase.

(C) An immunohistostaining was performed to study the expression ofMITF, DCT and tyrosinase proteins after 11 or 15 days of forskolinstimulation.

FIG. 6: Pharmacological WNT pathway activators increase WNT pathway inex vivo vitiligo skin and induce the upregulation of melanoblastmarkers.

Vitiligo patients (n=9) were biopsied in lesional skin area. Thebiopsies were stimulated in ex vivo culture during 14 days with GSK3βinhibitor: CHIR99021 (3 μM) or LiCl (20 μM) or with WNT agonist: SKL2001(40 μM) every other day. mRNA was extracted and a RT-qPCR was performedto quantify the relative expression of LEF (A) and the pre-melanocytesmarkers PAX3 (B), Brn2 (C) MITF (D) and DCT (E).

FIG. 7: Treatment of ex vivo depigmented skins from vitiligo patientswith pharmacological WNT pathway activators induces the differentiationof resident stem cells into pre-melanocytes. Depigmented skin fromvitiligo patients [A (control culture medium)] were stimulated in exvivo culture with WNT agonist SKL2001 (40 μM) (B), GSK3β inhibitorCHIR99021 (3 μM) (C) or GSK3β inhibitor LiCl 20 μM (D) every other dayto activate the WNT pathway. After 14 days, the skin response wasanalyzed by immunohistostaining with pre-melanocytes markers: a DCTstaining (green) and PAX3 staining (red). The co localization (yellow)observed on merge pictures show melanoblast in differentiation withinthe dermis.

FIG. 8. Schematic representation of factors involved in vitiligopathogenesis compared to healthy skin.

In healthy skin, the stimulation of the WNT pathway by keratinocytes andmelanocytes induces the differentiation and the proliferation ofmelanocyte stem cells allowing the constant turnover of the pools ofepidermal melanocytes. In vitiligo skin, the oxidative stress cantrigger the immune reaction in a genetically predisposed subject. Thedestruction of the melanocytes by the immune system releases melanocyteantigens that stimulate the auto-immune response and ultimately lead tothe complete disappearance of melanocyte from the epidermis (andsometimes the hair follicles). Concomitantly, the oxidative stressdecreases the WNT pathway activity in melanocytes and in keratinocytes.This induces an altered differentiation of melanocyte stem cells andthus alters the capacity of melanocyte turnover. Depending on thepatient and on the course of the disease, these two mechanisms aredifferentially implicated leading respectively to active depigmentationof the skin and resistance to repigmenting approaches.

FIG. 9:

(A) Schematic representation of skin sampling in vitiligo patient:Biopsies and Tape strippings were taken in lesional, perilesional andnon lesional area. Matched samples were obtained from a similar bodyarea from healthy volunteers.

(B) Characteristic of the studied population.

FIG. 10:

(A) NHM, (B) NHK and (C) whole skin in ex vivo culture were stimulated24 hours with 25 to 100 μM of H₂O₂. After mRNA extraction, a reversetranscription was performed and the relative gene expression of WNTfamily was analyzed with qPCR.

FIG. 11:

Vitiligo patients (n=9) were biopsied in lesional skin area. Thebiopsies were stimulated in ex vivo culture during 14 days with GSK3βinhibitor: CHIR99021 (3 μM) or LiCl (20 μM) or with WNT agonist: SKL2001(40 μM) every other day. mRNA was extracted and a RT-qPCR was performedto quantify the relative expression of WNT family.

EXPERIMENTAL PART

Vitiligo is an acquired depigmentation of the skin and sometimes hairfollicle affecting 0.5% to 1% of the world's population. It is clearlydemonstrated that vitiligo greatly impairs the quality of life ofaffected individuals and there is a strong therapeutic demand [Radtke,2009] [Silverberg, 2013]. The pathophysiology is complex and involvesmany cellular players. There are strong evidences for a role of bothoxidative stress and immune system in genetically predisposedindividuals [Passeron, 2012] [Spritz, 2012; Jin, 2012] [Bellei, 2013][Schallreuter, 2013]. One of the main problems studying vitiligo is thatthe affected cells, the melanocytes, are no longer present in thelesional skin. Moreover, increasing data emphasize the complexity of thepathophysiology of vitiligo with many cellular players involved such askeratinocytes, fibroblasts, stem cells, along with several types ofimmune cells. Thus, cell cultures can provide interesting informationbut they can't take into account the complex interactions occurring invitiligo skin. Recently, animal models using reactive T-cells againstmelanocyte antigens provided very interesting data on the immunereaction potentially involved in the depigmentation of vitiligo skin butthis model is not adapted to study mechanisms of melanocytesdifferentiation and repigmentation in vitiligo skin [Mosenson, 2013][Rashighi, 2014]. To date, there is no definitive treatment forvitiligo.

Interestingly, in current practice man can frequently observe that somevery active vitiligos can respond to treatment with a significantrepigmentation while the surrounding skin continues to depigment. On thecontrary, some patients with very stable vitiligo having no newdepigmentation for years cannot achieve any repigmentation even usingcombined therapeutic approaches. This strongly suggests that somemechanisms are involved in depigmenting the skin (and to the light ofthe current data the immune system appears to play a key role in thisprocess) while some others might be implicated to prevent or to inhibitthe lesional vitiligo skin from repigmenting. This hypothesis is alsosupported by the marked difference in the response to treatmentaccording to localizations. Indeed, hands, feet and face are the mostcommon affected areas in vitiligo and they are also often together thefirsts to depigment. However, although the face usually shows a highrate of response to treatment, hands and feet remain almost impossibleto repigment [Ostovari, 2004; Passeron, 2004].

In order to characterize the pathophysiological mechanisms implicated invitiligo human skin, inventors performed a transcriptome and proteomicanalysis on lesional, perilesional and non-depigmented skin of vitiligopatients compared to matched skin controls of healthy subjects. Whenlesional vitiligo skins were compared to healthy controls, 152 geneswere found to be upregulated and 181 down-regulated. Their results showthat the immune reaction occurs at very low-level but confirm thepotential role of CXCL10 for depigmenting vitiligo skin with asignificant increase in non-depigmented and perilesional vitiligo skincompared to healthy controls. However, neither CXCL10 nor other immunefactors were found to be deregulated in already depigmented vitiligoskin. Interestingly, the WNT pathway, implicated in melanocytesdifferentiation, was found to be altered in depigmented lesions but alsoin non-lesional skin. Inventors then showed in vitro that the oxidativestress decreases WNT expression/activation in keratinocytes and inmelanocytes. They developed an ex vivo skin model that remainsfunctional up to 15 days. They then confirmed the decreased activationof the WNT pathway in human skin subjected to oxidative stress. Finally,using pharmacological agents that activate the WNT pathway andpharmacological agents that inhibit GSK3B, they treated the ex vivodepigmented skins from vitiligo patients and success fully induced thedifferentiation of resident stem cells into pre-melanocytes. Theirresults shed light on the previously unrecognized role of a decreasedWNT activation in depigmented vitiligo skin preventing melanocytedifferentiation and they support further exploration of WNT agonists torepigment vitiligo lesions.

Material and Methods Patients for Transcriptomic Analysis

Ten patients with active non segmental vitiligo, defined by theoccurrence or the worsening of depigmented lesions in the past 3 monthswere included in the study after written consent was taken. The studywas approved by the local ethical committee. A 4 mm diameter skin biopsywas taken for each patient in the center of a vitiligo patch, in theperilesional area (defined by 5 mm outside the border of the lesion) andin non-lesional skin located in the same area but at 3 cm minimum of adepigmented lesion. A 4 mm biopsy was also taken and served as controlin 10 healthy patients matched for gender, age and localization. Ascheme explaining how the samples were taken and the characteristic ofthe population are described in FIGS. 9A and 9B.

Transcriptomic Analysis Quantification of Cytokines by Luminex inStratum Corneum.

Stratum corneum was collected by tape stripping using Dsquames®, (Ø2.2cm, D100, Monaderm, Monaco, France) as indicated in FIG. 9A. Proteinswere extracted from non-lesional, lesional and perilesional stratumcorneum in 2 ml Eppendorf tubes as follows: five Dsquames® were pooledand incubated in 200 μL ice-cold PBS containing Triton X100 0, 2% andprotease inhibitors (complete, Mini, EDTA-free from Roche AppliedScience, Mannheim, Germany) overnight at 4° C. under stirring at 1400rpm in a thermo mixer (Eppendorf, Hanbourg Germany). To remove insolublematerial, protein extracts were passed through 0.22 μm filters bycentrifugation (Ultra free-MC PVDF, Millipore, Billerica, Mass.).Protein concentrations were determined using Dc Protein Assay (DC Kit,Bio-Rad Laboratories, Hercules, Calif.) and serum albumin as a standard.Extracts were kept at −80° C. until used. Cytokines were quantified induplicate using following Luminex assays (Life Technologies, Carlsbad,Calif.): Human Cytokine, Premixed 62 Plex, ref PC1062M, ImmunoassayProcarta kit from Affymetrix (Santa Clara, Calif., USA). Qualitycontrols of all standard curves were performed. Cytokines werenormalized to the total concentration of protein. All protein extractswith a concentration lower than 0.15 mg/ml of total protein, were notretained for the quantification of cytokines. Limits of quantificationwere calculated and considered for each protein. For cytokines withvalues below the limit of quantification, these values were not retainedfor normalization (i.e. calculation of cytokine levels in pg/mg of totalprotein). Fold changes were calculated and statistical analysis wasperformed using Array studio, T TEST linear model, False Discovery Rate:0.05.

Cell Culture

NHMs and NHKs are obtained from the foreskin of phototype IV children asdescribed previously. Briefly, epidermal cells are obtained by overnightdigestion of the skin in a dispase solution (Roche) at 4° C. followed bythe digestion of the epidermis in a trypsin/EDTA solution during 20minutes at 37° C. NHMs are isolated in MCDB 153 medium (Sigma Aldrich)supplemented with 2% FBS (Perbio), 5 μg/ml insulin (Sigma Aldrich), 0.5μg/ml hydrocortisone (Sigma Aldrich), 16 nM TPA (Sigma Aldrich), 1 ng/mlFGF (Promega), 15 μg/ml Bovine Pituitary Extract (Invitrogen), and 10 μMForskolin (Sigma Aldrich) and 20 μg/ml geneticin (Invitrogen) during 2weeks.

NHKs are isolated in KGM 2 medium (Promocell).

All cells are maintained at 37° C. in a 5% CO₂ atmosphere.

Ex Vivo Skin Culture

Skin from abdominoplasty surgery was used to the development of the exvivo skin culture model. The subcutaneous fat was removed and biopsiesof 6 mm were done in the skin composed of dermis and epidermis.

For vitiligo skin biopsies, after verification of the absence ofmelanocytes with Wood's lamp, two or three 6 mm biopsies composed ofdermis plus epidermis were taken into lesional skin from vitiligopatients (n=9). The biopsies are rapidly put on transwell chamber of 0.4μm (Becton Dickinson) and kept on semi liquid culture conditions in“Skin long term culture medium” (Biopredic). The skin was maintained at37° C. in a wet 5% CO₂ atmosphere.

The culture medium supplemented with Forskolin (Sigma Aldrich), LiCl(Sigma Aldrich), CHIR99021 (Calbiochem) or SKL2001 (Calbiochem) waschanged every days during 14 days.

mRNA Extraction and Analysis

mRNA from melanocytes and keratinocytes was extracted with RNeasy kit(Qiagen). The biopsies are cut and disrupted with tissue ruptor (Qiagen)and the mRNA was extracted with RNeasy kit (Qiagen). cDNA weresynthesized from 300 ng to 1 μg of mRNA with the Reverse TranscriptionSystem (Promega). Relative quantification of gene expression wasmeasured by real time qPCR with Sybr green reagent (Life technology) onStep one system.

Infection of NHM and NHK and Luciferase Assay

NHK and NHM were co-infected with the lentivirus TCF/LEF ReporterLuciferase and Cignal Lenti Reporter Control Renilla (Qiagen) and theinfected cells were selected with Puromycin for 14 days.

NHM and NHK selected were stimulated 24 h with SKL2001 and H₂O₂ and theTCF/LEF activity was measured with Dual Luciferase Reporter Assay System(Promega).

Hematoxylin Eosin Staining

Biopsies are frozen in Tissu Tek OCT (Sakura), cut with cryostat in 7 μMsection and disposed on superforst plus slides (Thermoscientific).Briefly, after fixation with Paraformaldehyde 4% (EMS), the slide wasstained with Harry's Hematoxylin (Sigma Aldrich), rinse with chloridealcohol and carbonate Lithium (Sigma Aldrich) and stained withErythrosine (Sigma Aldrich). After several rinses, the slides weredehydrated with ethanol and xylene and fixed with mounting medium.

Immunohistofluorescence

Biopsies were fixed with 4% formol, embedded in paraffin, cut withmicrotome of 7 μM sections and fixed on superfrost plus slides(Thermoscientific). After deparaffinization by successive xylene andethanol incubations, the sections were hydrated with distillated water,permeabilized 10 minutes with 0.3% Triton and heated in microwave 12minutes in citrate buffer (Vector Laboratory) to unmasking antigen. Thesections were blocked 30 minutes in 10% goat serum and incubated inprimary antibodies MITF (Abcam), Tyrosinase (αPEP7h), DCT (Novus)overnight at 4° C. After rinsing with PBS 0.05% Tween, the sections wereincubated 1 hour with secondary antibodies Alexa 488 or Alexa 594(Invitrogen) and Hoechst (Invitrogen). After rinsing, the slides werefixed with mounting medium.

Results

Transcriptome Analysis Shows that Immune Reaction Occurs Only atLow-Level and is Restricted to Non-Depigmented and Perilesional VitiligoSkin and Reveals a Decreased Activation of the WNT Pathway Along withthe Absence of Immune Reaction Signature in Already Depigmented Skin

A total of 152 genes were found to be up-regulated and 181down-regulated when lesional vitiligo skins were compared to healthycontrols (FIG. 1A). When peri-lesional and non-depigmented vitiligoskins were compared to healthy controls, X, Y genes were found to beup-regulated and W, Z genes down-regulated, respectively. Interestingly,the ingenuity canonical pathway analysis revealed along with melanocytedevelopment and pigmentation signaling that the circadian signaling andthe WNT/beta-catenin pathway were significantly deregulated in vitiligolesions compared to control (FIG. 1B). The cluster analysis ofmicroarray data of the top genes from healthy skin (HS), non lesional(NLS), perilesional (PLS) and lesional skin (LS) from vitiligo subjectsshowed not surprisingly mostly melanocytic genes (FIG. 1C). Inventorsspecifically analyzed the expression of the genes related to the immunereaction. They observed very few variations among the several conditionsand none reach the level of statistical significance (see Table 1).

Table 1 HS NLS LS LS vs HS NLS vs HS PLS vs HS LS vs NLS PLS vs NLS CellPop Probeset Gene symbol Mean_Expressions FC P-val FC P-val FC P-val FCP-val FC P-val B cells 217281_x_at IGH@ 88 78 82 0.9 0.9 0.7 0.8 1.0228518_at IGHG1/IGHM 70 64 67 0.6 0.3 0.6 0.5 0.9 216379_x_at CD24 81897213 7343 0.4 0.4 0.3 0.8 1.0 205297_s_at CD79B 126 106 102 0.2 0.3 0.30.7 1.0 211693_at IGHA1 135 125 133 0.9 0.4 1.0 0.4 0.9 223502_s_atTNFSF13B 265 296 240 0.7 0.7 0.9 0.2 0.9 Langerhans 210325_at CD1A 10961267 1354 0.2 0.5 0.5 0.5 1.0 cells 211734_s_at FCER1A 2992 3198 33180.6 0.8 0.9 0.7 0.9 204232_at FCER1G 1480 1465 1492 1.0 1.0 0.8 0.9 0.9207496_at MS4A2 147 137 150 1.0 0.9 0.8 0.6 1.0 220428_at CD207 629 713739 0.5 0.7 0.8 0.8 0.9 DermaIDCs 206749_at CD1B 56 103 70 0.7 0.2 0.50.2 0.9 immature 205987_at CD1C 825 992 822 1.0 0.6 1.0 0.3 0.9207277_at CD209 792 926 821 0.9 0.7 0.9 0.6 0.9 210184_at ITGAX 106 11293 0.6 0.9 0.6 0.2 0.9 205569_at LAMP3 934 1324 1322 0.4 0.4 0.3 1.0 1.0200785_s_at LRP1 1377 1369 1195 0.6 1.0 0.6 0.3 0.9 205668_at LY75 7211026 983 0.1 0.1 0.2 0.7 0.9 204438_at MRC1 3259 3029 2761 0.5 0.8 0.50.5 0.9 204924_at TLR2 133 135 108 0.4 1.0 0.8 0.1 0.9 221060_s_at TLR4310 268 287 0.9 0.8 0.9 0.8 1.0 DermaIDCs 205987_at CD1C 825 992 822 1.00.6 1.0 0.3 0.9 activated 200784_s_at LRP1 464 454 418 0.6 0.9 0.6 0.50.9 210325_at CD1A 1096 1267 1354 0.2 0.5 0.5 0.5 1.0 204440_at CD83 366452 428 0.6 0.5 0.6 0.7 0.9 205686_s_at CD86 153 145 148 0.9 0.9 0.8 0.91.0 217028_at CXCR4 3300 2857 2296 0.1 0.7 0.5 0.3 0.9 pDCs 206148_atIL3RA 130 118 114 0.3 0.5 0.2 0.7 0.9 pDCs 211734_s_at FCER1A 2992 31983318 0.6 0.8 0.9 0.7 0.9 204232_at FCER1G 1480 1465 1492 1.0 1.0 0.8 0.90.9 207496_at MS4A2 147 137 150 1.0 0.9 0.8 0.6 1.0 203547_at CD4 423395 354 0.4 0.8 0.4 0.4 0.9 212298_at NRP1 623 632 639 0.9 1.0 0.9 0.91.0 220146_at TLR7 134 132 134 1.0 1.0 0.8 1.0 0.9 229560_at TLR8 116109 109 0.9 0.9 0.9 1.0 1.0 IDECs 204438_at MRC1 3259 3029 2761 0.5 0.80.5 0.5 0.9 210325_at CD1A 1096 1267 1354 0.2 0.5 0.5 0.5 1.0 206749_atCD1B 56 103 70 0.7 0.2 0.5 0.2 0.9 205987_at CD1C 825 992 822 1.0 0.61.0 0.3 0.9 211734_s_at FCER1A 2992 3198 3318 0.6 0.8 0.9 0.7 0.9204232_at FCER1G 1480 1465 1492 1.0 1.0 0.8 0.9 0.9 207496_at MS4A2 147137 150 1.0 0.9 0.8 0.6 1.0 205786_s_at ITGAM 460 421 446 1.0 0.9 1.00.8 0.9 242197_x_at CD36 298 324 203 0.3 0.9 0.7 0.0 0.9 209555_s_atCD36 1392 998 811 0.3 0.6 0.6 0.5 1.0 221463_at CCL24 66 58 64 0.8 0.40.7 0.3 0.9 1555759_a_at CCL5 225 236 219 1.0 0.9 0.9 0.8 1.0211734_s_at FCER1A 2992 3198 3318 0.6 0.8 0.9 0.7 0.9 204232_at FCER1G1480 1465 1492 1.0 1.0 0.8 0.9 0.9 207496_at MS4A2 147 137 150 1.0 0.90.8 0.6 1.0 Mastcells 216474_x_at TPSAB1/TPSB2 3027 3215 3397 0.8 0.91.0 0.8 0.9 214533_at CMA1 665 608 725 0.8 0.9 0.9 0.3 1.0 205683_x_atTPSAB1 3123 3329 3533 0.7 0.9 1.0 0.8 1.0 207134_x_at TPSB2 2558 27432912 0.7 0.9 1.0 0.8 0.9 214568_at TPSD1 74 72 77 0.7 0.9 1.0 0.4 0.9220339_s_at TPSG1 62 65 70 0.4 0.8 0.8 0.6 1.0 Neutrophils 206871_atELANE 60 81 81 0.8 0.8 0.8 1.0 0.9 202018_s_at LTF 421 268 164 0.3 0.70.2 0.5 0.9 1555349_a_at ITG82 284 295 254 0.7 0.9 0.8 0.3 0.9Macrophages 201743_at CD14 864 873 792 0.8 1.0 0.8 0.6 0.9 215049_x_atCD163 1145 1398 1275 0.8 0.6 0.9 0.7 0.9 203507_at CD68 96 72 78 0.6 0.40.4 0.4 1.0 205786_s_at ITGAM 460 421 446 1.0 0.9 1.0 0.8 0.9202803_s_at ITG62 504 543 448 0.7 0.9 0.8 0.2 0.9 T cells 217326_x_atIL23A/TRBV19 93 91 92 0.9 0.8 0.2 0.9 0.9 216920_s_at TARP/TRGC2 78 10894 0.8 0.6 0.5 0.7 1.0 210972_x_at TRAC/TRAJ17 653 644 541 0.5 1.0 0.90.2 0.9 206545_at CD28 73 70 60 0.6 0.9 0.5 0.6 0.9 213539_at CD3D 338398 322 0.9 0.7 1.0 0.3 0.9 206804_at CD3G 84 94 75 0.8 0.8 0.9 0.3 0.9203547_at CD4 423 395 354 0.4 0.8 0.4 0.4 0.9 205758_at CD8A 279 290 2300.6 1.0 1.0 0.3 1.0 236341_at CTLA4 61 95 76 0.7 0.4 0.5 0.4 0.9214470_at KLRB1 175 202 156 0.8 0.8 1.0 0.2 0.9 209670_at TRAC 723 686606 0.4 0.9 0.7 0.4 1.0 211796_s_at TRBC1 816 902 676 0.6 0.9 0.9 0.20.9 216191_s_at TRDV3 143 119 130 0.9 0.8 0.9 0.8 0.9 211902_x_at YME1L1670 663 542 0.5 1.0 0.9 0.2 1.0 Th1 206295_at_ IL18 3261 2595 2983 0.70.2 0.4 0.1 0.9 AFFX-HUMISGF3A/ STAT1 220 305 276 0.5 0.3 0.2 0.7 0.9M97935_M8_at AFFX-HUMISGF3A/ STAT1 1655 1904 1751 0.9 0.6 0.5 0.6 1.0M97935_3_at 206118_at STAT4 104 101 83 0.3 0.9 0.3 0.3 0.9 Cell HS NLSLS LS vs HS NLS vs HS PLS vs HS LS vs NLS PLS vs NLS Population ProbesetGene symbol Mean_Expressions FC P-val FC P-val FC P-val FC P-val FCP-val 207113_s_at TNF 86 115 110 0.2 0.1 0.4 0.8 0.9 206890_at IL12RB163 59 66 0.7 0.5 0.9 0.1 0.9 Th17 203085_s_at TGFB1 163 170 175 0.8 0.91.0 0.8 0.9 208991_at STAT3 4197 4454 4173 1.0 0.7 0.7 0.3 1.0 225289_atSTAT3 932 947 973 0.9 1.0 0.7 0.8 0.9 228806_at RORC 232 289 271 0.3 0.20.2 0.4 1.0 207113_s_at TNF 86 115 110 0.2 0.1 0.4 0.8 0.9 206983_atCCR6 516 468 475 0.7 0.7 0.7 0.9 1.0 Th2 201331_s_at STAT6 3645 27562886 0.1 0.1 0.1 0.6 1.0 203233_at IL4R 562 585 510 0.8 0.9 0.6 0.4 0.9Treg 224211_at FOXP3 78 82 79 0.9 0.8 0.8 0.7 1.0 Chemokines 205392_s_atCCL14/CCL14− 3178 2449 2232 0.2 0.5 0.2 0.4 0.9 205114_s_at CCL15/CCL15/75 67 68 0.8 0.9 0.8 1.0 1.0 CCL3/CCL3L1/ CCL3L3/ 206407_s_at CCL13 18993519 2504 0.4 0.04 0.3 0.2 0.9 32128_at CCL18 565 1512 922 0.50 0.1 0.40.2 0.9 210072_at CCL19 588 664 459 0.6 0.9 0.7 0.2 0.9 216598_s_at CCL2878 1016 957 0.9 0.8 1.0 0.8 0.9 204606_at CCL21 1895 1989 1773 0.9 0.91.0 0.4 0.9 207861_at CCL22 430 584 538 0.6 0.4 0.6 0.7 0.9 210549_s_atCCL23 133 103 113 0.8 0.7 0.7 0.4 0.9 207955_at CCL27 3493 4217 3880 0.90.8 0.9 0.7 0.9 1555759_a_at CCL5 225 236 219 1.0 0.9 0.9 0.8 1.0214038_at CCL8 292 382 249 0.8 0.7 1.0 0.3 0.9 203915_at CXCL9 94 163189 0.6 0.8 0.6 0.9 1.0 823_at CX3CL1 213 249 212 1.0 0.7 0.9 0.4 0.9206366_x_at XCL1 87 93 100 0.7 0.9 0.7 0.8 0.9 Chemokine 205098_at CCR1143 147 151 0.9 1.0 1.0 0.9 1.0 receptors 206978_at CCR2 178 187 159 0.80.9 0.8 0.5 0.9 211286_x_at CSF2RA 112 109 110 0.9 0.9 0.7 0.9 0.9205159_at CSF2RB 530 709 651 0.3 0.1 0.2 0.5 1.0 205898_at CX3CR1 277278 266 0.9 1.0 0.9 0.8 0.9 207008_at CXCR2 170 130 148 0.7 0.4 0.4 0.51.0 217028_at CXCR4 3300 2857 2296 0.1 0.7 0.5 0.3 0.9 Cytokines205992_s_at IL15 104 142 126 0.6 0.4 0.5 0.5 0.9 227401_at IL17D 450 507443 1.0 0.8 0.8 0.4 0.9 203828_s_at IL32 768 783 630 0.6 1.0 0.7 0.2 0.9209821_at IL33 1310 940 928 0.1 0.2 0.1 0.9 0.9 224555_x_at IL37 49094131 4379 0.6 0.5 0.7 0.6 0.9 231755_at IL36B 134 81 105 0.5 0.1 0.3 0.10.9 220322_at IL36G 543 460 690 0.6 0.8 1.0 0.1 0.9 222223_s_at IL36RN3045 2873 3793 0.3 0.9 0.8 0.0 0.9 206693_at IL7 177 171 212 0.4 0.9 0.70.0 0.9 212195_at IL6ST 3950 3922 3728 0.6 1.0 0.7 0.4 0.9 207339_s_atLTB 171 225 188 0.8 0.3 0.7 0.3 0.9 223501_at TNFSF13B 144 159 144 1.00.7 1.0 0.5 0.9 Cytokine 202727_s_at IFNGR1 3317 3291 3211 0.8 1.0 0.70.7 0.9 receptors 1552584_at IL12RB1 225 267 253 0.8 0.7 0.7 0.9 1.0201887_at IL13RA1 1440 1598 1433 1.0 0.6 0.8 0.3 0.9 207375_s_at IL15RA267 274 259 0.9 1.0 0.8 0.7 0.9 219115_s_at IL20RA 732 812 867 0.5 0.70.5 0.6 0.9 205926_at IL27RA 88 83 71 0.4 0.9 0.5 0.3 0.9 205291_atIL2RB 223 161 162 0.2 0.3 0.3 1.0 1.0 204116_at IL2RG 410 453 383 0.90.9 1.0 0.3 0.9 203233_at IL4R 562 585 510 0.8 0.9 0.6 0.4 0.9 226218_atIL7R 550 458 327 0.2 0.8 0.5 0.1 0.9 226621_at OSMR 1317 1370 1332 1.00.9 1.0 0.7 0.9

Interestingly, a transcriptional network linking WNT signaling andmelanogenesis pathways was observed using inguinity analysis pathways(FIG. 2). Lesional vitiligo skin is both characterized by adown-regulated expression of key transducer of WNT signaling pathway(E.g. LEF1), and by the upregulation of negative regulators of WNTsignaling pathway DVL1 and p53 (involved in signal transduction of WNTmembers) and TLE4 (groucho family member) and ZBTB33/Kaiso (involved intranscriptional repression of WNT target genes).

The circadian genes were found to be modulated in our transcriptionalanalysis. Due to the demonstrated role of the variation of these genesduring the day, inventors investigate if this modulation was due to thetime when the skin samples were biopsied or was linked to vitiligopathogenesis. They thus analyzed the expression of the key circadiangenes (ARNTL (Bmal1), CLOCK, PER1, NR1D1) accordingly to the hour of thesamples were taken. A strong correlation between the hours of thesamples and the expression of all these genes was found, showing thatthe modulation of the circadian gene expression was not linked to thevitiligo but to the moment where the samples were taken. The results ofthe transcriptional analysis were then controlled using TLDA. The markeddecrease in all the melanocytic genes in vitiligo skin compared tocontrol confirms the loss of melanocytes in the affected vitiligo skin(FIG. 3A). Inventors then analyzed the level of expression of LEF1 as akey marker of the activation of the WNT pathway. Conformingly with theresults of the transcriptional analysis, the expression of LEF1 wasfound to be down-regulated in lesional vitiligo skin (FIG. 3B). As anadditional control and to investigate if the expression of LEF1 could bemodulated by the circadian cycle, they synchronized melanocytes andfollowed the relative expression of ARNTL and LEF1. The results showedthat the expression of LEF1 is not correlated with the expression ofANRTL, a marker of circadian cycle. They reproduced this experimentfollowing the expression of several WNT mRNA during the circadian cycleand observed no correlation (data not shown). Thus, the modulations ofthe WNT pathway observed in the transcriptome and TLDA analyses arelinked to the pathogenesis of vitiligo. Finally, taking into account theresults that suggested in mice model of vitiligo the role of CXCL10,they also analyzed its expression in the skin samples by TLDA.Interestingly, they observed a significant increase in mRNA expressionof CXCL10 in perilesional skin but also in non-depigmented skin ofvitiligo patients compared to healthy controls. However, the level ofexpression of CXCL10 was not different from healthy skin in depigmentedvitiligo lesions (FIG. 3C).

Taken together, these results obtained from vitiligo patients comparedto healthy subjects do not show significant modulation of genes involvedin the immune reaction except for CXCL10 that was found to beup-regulated in peri-lesional but also non-depigmented skin of vitiligopatients. They also show that no more immune reaction is observable inlesional skin devoid of melanocytes. Interestingly, they mostly reveal adown-regulation of the WNT/beta-catenin pathway in vitiligo skin.

Analysis of Stratum Corneum Cytokines Shows No Significant Dysregulationin Vitiligo Skin

The concentrations of 62 cytokines were measured in stratum corneum ofsubject with vitiligo and healthy volunteers using tape stripping aminimal invasive method. Twelve cytokines were detected and quantifiedfrom stratum corneum extracts with a concentration ranging from 1 to7000 pg/mg of protein (see below Table 2).

TABLE 2 Quantity of cytokines in stratum corneum cytokines HealthyLesional Non-lesional Peri-lesional IL-1RA (IL1R1) 5626 7012 3201 7058IL-1alpha (IL-1F1) 2245 3165 3242 3499 G-CSF (CSF-3) 1987 1812 995 1850EGF 37 17 9 9 CXCL10 (IP-10) 23 38 34 35 CXCL9 (MIG) 19 26 24 24IL-1Beta (IL-1F2) 15 30 8 30 sICAM-1 (CD54) 12 14 10 10 sVCAM-1 (CD106)8 7 5 6 CCL11 (EOTAXIN ) 7 8 7 7 CXCL1 (GROalpha) 6 6 6 6 CCL2 (MCP-1) 55 4 4

Quantity (pg/mg) of cytokine in stratum corneum (meannormalized values).sFAS TNFRSF6, GM-CSF CSF-2, MCP-3 CCL7, IL-15, TNF-alpha, sE-SelectinCD62E ELAM1, MPO Myeloperoxidase, VEGF-A, IL-22 IL-TIF, MIP1-Beta CCL4,IL-3, IL-27, IL-4, ENA-78 CXCL5, SDF-1 CXCL12, Trail TNFSF10, IL-5,IL-20, FRACTALKINE CX3CL1, GRANZYME B, IFNomega IFNW1, IL-8 CXCL8,MIP-3alpha CCL20, IL-10 CSIF, IFNalpha2, BLC CXCL13 BCA-1, IL-7, PDGFBB,I-TAC CXCL11 , IL-17F ML-1, IL-17A CTLA8, IL-6, sCD40-Ligand TNFSF5CD40LG, TNF-Beta TNFSF1 LTA, IL-13, IL-23 p19, IFNg, IL-12 p′70, IL-21,IL-2, IL-9, TNFRI TNFRSF1A, TNFR II TNFRSF1B, RANTES CCL5, TGFalpha,IL-2RA CD25, IFNbeta, FGF BASIC, IL-12 IL-23 p40, MIP-1 alpha CCL3 werefound under limit of quantification.

No significant difference in cytokine profile between pathological andnon-pathological samples was observed (Table 3).

The concentration of CXCL10 protein in the stratum corneum was comparedto the mRNA expression obtained with the skin biopsies. While CXCL10 wasfound to be significantly up-regulated within the non lesional and perilesional vitiligo skins compared to controls at the mRNA level, nosignificant modulation was observed at the protein level in the stratumcorneum (see below Table 4).

These results show that the immune reaction in vitiligo occurs at verylow-level.

Oxidative Stress Decreases WNT Pathway in the Skin

Since the WNT pathway, implicated in melanocytes differentiation, seemsto be affected in vitiligo skin, inventors tried to determine theelement—known to be involved in vitiligo—responsible for thisdysregulation. Oxidative stress was reported to inhibit theWNT/beta-catenin pathway in kidney cells [Shin, 2004]. Inventors studiedthe impact of oxidative stress on WNT/beta-catenin activation within theskin. They induced an oxidative stress with H₂O₂ in melanocytes andkeratinocytes and analyzed the expression of LEF and WNT family. After24 hours in presence of H₂O₂, the expression of LEF and of the majorityof WNT was decreased both in melanocytes (FIG. 4A and FIG. 10A) andkeratinocytes (FIG. 4B and FIG. 10B). The same result was observed inwhole skin as the stimulation of skin biopsies in an ex-vivo model(validated in FIG. 4) with H₂O₂ induced a decrease expression of LEF andof most of the WNT family (FIG. 4C and FIG. 10C).

Finally, inventors studied the LEF promoter activity under oxidativestress. They used a TCF/LEF luciferase reporter in melanocytes andkeratinocytes. Concordantly to the decrease expression of several WNTunder oxidative stress, the activity of the TCF/LEF promoter was alsodecreased in a dose responsive manner under oxidative stress.

Taken together these results show that the oxidative stress maintains alow activation of the WNT/beta-catenin pathway.

Development of an Ex Vivo Model to Study Vitiligo Skin

The difficulty to study vitiligo disease is due in particular to theabsence of model that mimics the in vivo conditions and also containsstem cells that could be targeted to induce repigmentation. Inventorsdeveloped an ex vivo skin model viable for a period long enough to allowinduction of the differentiation of melanocyte stem cells in vitiligoskin. In clinical practice vitiligo lesions usually take months toachieve repigmentation (if any). However, in best cases, the onset ofpigmentation can sometimes be observed after 15 days of treatment.Inventor's objective was to obtain a skin morphologically correct andable to respond to a pigmentation inducer, such as forskolin, after 15days of culture ex vivo. From abdominoplasty skin surgery, inventorsdetermine the conditions required to satisfy these conditions. They used6 mm biopsies composed of dermis and epidermis, disposed on transwellchamber in order to put the skin in semi liquid culture conditions.After 15 days in ex vivo culture, the morphology of the skin wasconsidered acceptable. They noted only a decrease of dermal papillaecompared to initial conditions that could be due to a difference oftension in the skin (FIG. 5A). The capacity of the skin to responds toforskolin was then analyzed by qPCR and immunohistofluorescence bystudying the expression of the melanogenic genes MITF, DCT andtyrosinase. A dose response of forskolin was performed with onestimulation of forskolin in systemic conditions every other day. Asexpected, the first response was the up-regulation of MITF mRNA (FIG.5B) and protein (FIG. 5C) after 11 days of stimulation. This phenomenonis transient but a high expression persists after 15 days at lowforskolin concentration (FIG. 5B). At 15 days, they observed a strongincrease at mRNA and protein level of the melanogenic enzymes DCT andtyrosinase (FIGS. 5B and 5C). To conclude, inventors obtained a model ofex vivo skin culture viable and functional until 15 days of culture.

Pharmacological WNT Pathway Activators Induce an Increase of WNTExpression in Ex Vivo Vitiligo Skin.

WNT pathway is implicated in melanocyte differentiation and inventorsshowed that it is altered in vitiligo skin. In order to induce thedifferentiation of melanocyte stem cells, they pharmacologicallyactivated the WNT pathway in vitiligo skin cultured ex vivo as shownpreviously. They used two methods to activate the WNT pathway: a WNTagonist (SKL2001) and two GSK3β inhibitors: lithium chloride (LiCl) anda commercial specific inhibitor (CHIR99021). They biopsied 9 vitiligosubjects (6 mm punch) on diverse body locations (4 on elbow, 2 on trunk,2 on leg, 1 on arm and 1 on axilla) and treated the ex vivo culturemodel during 14 days with a systemic stimulation every other day withthe WNT activators.

They evaluated the activation of WNT pathway by the mRNA level of WNTs(TCF/LEF1 and the melanogenesis pathway by the mRNA level of MITF, DCT,PAX3 and BRn2) within the skin. Treatments with LiCl, CHIR99021 andSKL2001 induce an increase of all WNT and LEF after 14 days ofstimulation (FIG. 6A and FIG. 11). In their model the LiCl wasparticularly efficient to up-regulate WNT expression.

Treatment of Ex Vivo Depigmented Skins from Vitiligo Patients Inducesthe Differentiation of Resident Stem Cells into Pre-Melanocytes.

Since inventors succeeded to increase the WNT pathway in the ex vivovitiligo skins, they analyzed the expression of melanoblast markersunder treatment. The mRNA level of early melanoblast markers as PAX3 andBrn2 were up-regulated in biopsies stimulated with CHIR99021, SKL2001and LiCl (FIGS. 6B and 6C). This result suggests an initiation ofmelanocytes differentiation. The level of MITF was increased only inskin treated with SKL2001 but its expression is transient afterstimulation so they supposed that the time response was differentbetween the treatments (FIG. 6D). The pre-melanocyte marker DCT wasincreased in response to all treatments but the effect was stronger inresponse to LiCl (FIG. 6E).

As they observed the up-regulation of mRNA level of melanoblast markersin the skin treated with WNT pathway activators, they investigated ifthese treatments have allowed the differentiation of melanocytes stemcells in pre-melanocytes in the skin by studying the co-expression ofDCT and PAX3 by immunofluorescence. In vitiligo skins cultured incontrol conditions, they observe no or few isolated cells expressingPAX3 and DCT (FIG. 7A). In stimulated conditions (FIGS. 7B, 7C, 7D),inventors found, in the dermis and in the hair follicles, many group ofcells expressing the two markers representing the melanocytes indifferentiation. Taken together these results showed that targeting thedeficient WNT pathway of vitiligo skin by using WNT agonists or GSK3Binhibitors allows the differentiation of melanocyte stem cells intopre-melanocytes.

Discussion

Inventors' results emphasize the complexity of vitiligo pathophysiology.While they support the role of the immune system and especially CXCL10for depigmenting the skin of vitiligo patients, they also show thatneither CXCL10 nor other immune factors are deregulated in alreadydepigmented vitiligo skin. Recently, the role of CXCL10 and the IFNγpathway was demonstrated in vitiligo mice model [Harris, 2012][Rashighi, 2014]. The increased expression of CXCL10 was also found tobe up-regulated in vitiligo skin. Interestingly, the skin samplesanalyzed were selected to still have an immune infiltrate [Rashighi,2014]. Inventor's results are consistent with these data as they show anincrease in CXCL10 expression in perilesional skin when compared to theCXCL10 expression in healthy control. However they also discovered thatCXCL10 expression is significantly increased within unaffected skin ofvitiligo patients when compared to healthy control skin, suggesting thateven non-depigmented skin in vitiligo patients has low-level immuneactivation that culminates in CXCL10 expression. On the contrary, theydo not observed an increased expression of CXCL10 in depigmented lesionsthat lack melanocytes, suggesting that the low-level immune activationin vitiligo skin is dependent on melanocytes. Other data support thefact that the immune reaction in vitiligo is restricted todifferentiated melanocytes [Mosenson, 2013] [Chatterjee, 2014]. The onlyother transcriptome analysis performed in vitiligo skin did not show apotent activation of the immune system and only reported an activationof some factors involved in the innate immunity (in particular onnatural killer cells) that inventors couldn't find in their analysis[Yu, 2012]. This first transcriptome study was performed in unaffectedskin and lesional depigmented areas and compared to matched control.Interestingly, the authors found also an activation of natural killercells in the unaffected vitiligo skin supporting a low-level immuneactivation in non-depigmented skin. They additionally performed aproteomic analysis of stratum corneum cytokines. The levels of cytokinesin stratum corneum using immunoassays had never been performed before invitiligo skin but they were investigated in several skin diseasesincluding for example psoriasis [Gearing, 1990] and atopic dermatitis[Kezic, 2012] and also in skins exposed to UVB [Janssens, 2009] orchemical agent such as sodium lauryl sulphate [de Jongh, 2007].Inventors found no significant modulation of cytokines between lesional,perilesional and unaffected vitiligo skin when compared to control. Thisis probably explained by the low-grade inflammation process occurring invitiligo as compared to other dermatitis such as psoriasis or atopicdermatitis. Interestingly, inventors recently showed in a prospectiverandomized placebo controlled study that twice weekly application of0.1% tacrolimus ointment significantly decreases the relapse rate ofvitiligo lesions that were successfully repigmented (Cavalié, J InvestDerm in Press). The efficacy of this maintenance therapy, previouslyalso shown in atopic dermatitis [Schmitt, 2011], strongly suggests inaccordance to the data gathered in the present transcriptional study,that a low-level immune reaction occurred in pigmented skin of vitiligopatients. To summarize, these data demonstrate in Human skin the role ofan immune reaction restricted to differentiate melanocytes and occurringat low-level in active vitiligo lesions but also in non-depigmentedvitiligo skin with a central role of CXCL10. On the contrary, the immunereaction is no longer present in vitiligo lesions devoid ofdifferentiated melanocytes.

Though, some depigmented vitiligo lesions will rapidly respond totreatment when other should resist to all the current approachesincluding phototherapy. Phototherapy is one of the key treatmentsagainst vitiligo [Taieb, 2013]. Its mechanism of action on vitiligo isstill poorly understood but more than its action on the immune reaction,phototherapy is mainly active through the stimulation of thedifferentiation of melanocyte stem cells. Interestingly, recent datashowed that the WNT/beta-catenin pathway is playing a key role in theUVB induced melanocyte stem cell differentiation [Yamada, 2013]. Withinthe skin, the secretion of WNT principally by keratinocytes contributesto the differentiation of stem cells in melanocytes. Inventors'transcriptional analysis reveals an alteration of the WNT/beta-cateninpathway in vitiligo skin with a significant decrease of LEF/TCFexpression that was confirmed using TLDA. They additionally showed thatoxidative stress inhibits the WNT/beta catenin activation in melanocytesand in keratinocytes. They confirmed this action on ex vivo skin models.Recently, a first link was discovered connecting oxidative stress andactivation of the immune response [Toosi, 2012] [Passeron, 2012]. Ourdata underline that the oxidative stress has also a negative impact onthe differentiation of melanocyte stem cells by inhibiting theWNT/beta-catenin pathway. Interestingly, several studies reported abeneficial role of oral or topical antioxidants for treating vitiligo[Schallreuter, 1995] [Dell'Anna, 2007] [Schallreuter, 2013]. However,there are some conflicting results on the efficacy of antioxidants fortreating vitiligo [Bakis-Petsoglou, 2009]. Thus, inventors furtherdecided to directly address the defective differentiation of melanocytestem cells by stimulating the WNT/beta-catenin pathway that theydemonstrated to be inhibited in vitiligo lesions. To this respect theydeveloped an ex vivo skin model that they show to be advantageouslyviable and functional to up to 15 days. Using this ex vivo model indepigmented skins of vitiligo patients they demonstrated that treatmentswith WNT agonists or GSK3B inhibitors induce a strong increase ofmelanocyte markers. Ultimately, they showed that such treatments inducethe differentiation of resident melanocyte stem cells in pre-melanocytesexpressing PAX3 and DCT. Interestingly, they observed pre-melanocytes inthe hair-follicles but also in the dermis suggesting that this approachcould be helpful to differentiate the dermal stem cells of glabrous skinthat are usually not effectively stimulated by phototherapy as aperifollicular pigmentation is observed in most cases. This allows arepigmentation of usually resistant areas such as hand or foot glabrousskin. Our results show the usefulness of ex vivo model of vitiligo skinsfor studying mechanisms involved in the differentiation of melanocytesbut also for testing new treatments. Beyond the development of a newmodel for studying vitiligo, these results emphasize the interest ofactivating the WNT/beta-catenin pathway for inducing differentiation ofmelanocyte stem cells and thus a repigmentation in vitiligo lesions. Thepreviously unrecognized implication of a decreased WNT/beta-cateninactivation in vitiligo lesions brings new clues for understanding thediscrepancy between therapeutic responses between localizations such asface compared to hands or feet. Indeed fibroblast-secreted factors, suchas DKK1, decrease pigmentation on palms and soles by inhibiting theWNT/beta-catenin pathway [Yamaguchi, 2008; Yamaguchi, 2007]. Increasingevidences show that dermal fibroblast characteristics define regionaldifferences in skin [Thangapazham, 2014]. To the light of our data, itcan be hypothesized that regional factors decreasing theWNT-beta-catenin pathway, such as DKK1, further inhibit the alreadydefective activation of this pathway in vitiligo skin and thus preventrepigmentation in these areas. The use of topical WNT agonist agentsthus represent a powerful approach for inducing repigmentation invitiligo skin that might be also effective in difficult to treat areassuch as hands and feet. For repigmenting vitiligo lesions, a topicalapproach would be sufficient and safe. Topical agents activating theWNT-beta-catenin pathway such as lithium are already used indermatological practice [Kastarinen, 2014]. Inventors herein reveal thatan adapted formulation allowing effective concentration of lithium inthe lower layers of the epidermis but also in the dermis is an effectiveapproach to stimulate the differentiation of melanocytes obtained in theex vivo model using lithium treatment as herein described.

Taken together, inventors results show that the immune reaction invitiligo occurs only at very low-level, in particular with an increasedCXCL10 expression in non-depigmented skin and in peri lesional skin,while immune reaction is no longer detectable in vitiligo lesion withoutremaining melanocytes.

They also shed light on the previously unrecognized defect onWNT/beta-catenin activation triggered by oxidative stress that preventsthe differentiation of melanocyte stem cells (FIG. 8). In addition tothe better understanding of the complex pathophysiology of vitiligo,these results provide new therapeutic options to be confirmed inclinical trials.

REFERENCES

Bakis-Petsoglou, 2009

Bellei, 2013

Benson N R, et al., “An analysis of select pathogenic messages inlesional and non-lesional psoriatic skin using non-invasive tapeharvesting”. J Invest Dermatol. 2006 October; 126(10): 2234-41

Cavalié, J Invest Derm in Press

Chatterjee, 2014

De Jongh, 2007

Dell'Anna, 2007

Gearing, 1990

Harlow et al. (Antibodies: A laboratory Manual, CSH Press, 1988)

Harris, 2012

Janssens, 2009

Jin, 2012

Kastarinen, 2014

Kezic, 2012

Kohler et al. (Nature 256 (1975) 495

Lepe, 2003

Mosenson, 2013

Ongenae K et al. 2006

Ortonne J P 2005

Ostovari, 2004

Passeron, 2004

Passeron, 2012

Radtke, 2009

Rashighi, 2014

Schallreuter, 1995

Schallreuter, 2013

Shin, 2004

Silverberg, 2013

Schmitt, 2011

Spritz 2007

Spritz, 2012

Taieb, 2013

Thangapazham, 2014

Toosi, 2012

Ward et al. (Nature 341 (1989) 544)

Wong R et al., “Use of RT-PCR and DNA microarrays to characterize RNArecovered by non-invasive tape harvesting of normal and inflamed skin”.J Invest Dermatol. 2004 July; 123(1):159-67

Wong R et al., “Analysis of RNA recovery and gene expression in theepidermis using non-invasive tape stripping”; J Dermatol Sci. 2006November; 44(2):81-92

Yamada, 2013

Yamaguchi, 2007

Yamaguchi, 2008

Yu, 2012

1. A method for treating a hypopigmentation disorder and/or repigmentinga hypopigmentation lesion in a subject, comprising administering to thesubject a therapeutically effective amount of an activator of at leastone protein of the WNT signalling pathway.
 2. The method according toclaim 1, wherein the hypopigmentation disorder is vitiligo.
 3. Themethod according to claim 1, wherein the at least one protein of the WNTsignaling pathway is selected from LEF (Lymphoid enhancer-bindingfactor), TCF (DNA-binding transcription factor), MITF(Microphthalmia-associated transcription factor), DCT (Dopachrometautomerase or dopachrome delta-isomerase, tyrosine-related protein 2),PAX3 (transcription factor 3 of the paired box (PAX) family) and BRn2transcription factor. 4-7. (canceled)
 8. A method for the in vitro or exvivo detection, diagnosis or staging of a hypopigmentation disorder in asubject suspected of suffering of a pigmentation disorder, comprisinganalysing the expression of at least one protein of the WNT signalingpathway in a biological sample from the subject, said analysis providinginformation on the presence or stage of a hypopigmentation disorder inthe subject.
 9. The method of claim 8, wherein the analysis comprisesdetermining the presence, absence or amount of at least one protein ofthe WNT signaling pathway, the presence of the at least one protein ofthe WNT signaling pathway or an expression thereof lower than areference amount being indicative of the presence or stage of ahypopigmentation disorder in the subject.
 10. The method of claim 8,wherein analysing at least one protein of the WNT signaling pathwaycomprises contacting a sample, or an aliquot thereof, with a specificbinding reagent that binds the at least one protein of the WNT signalingpathway or the nucleic acid encoding said protein and determining thepresence or amount of the at least one protein of the WNT signalingpathway or of the nucleic acid encoding said protein bound to saidbinding reagent.
 11. The method of claim 10, wherein the binding reagentis selected from a complementary nucleic acid, an antibody, and afragment or derivative thereof.
 12. (canceled)
 13. A kit comprising adevice comprising at least one complementary nucleic acid, antibody,fragment or derivative thereof that binds at least one protein of theWNT signaling pathway or a nucleic acid encoding said at least oneprotein of the WNT signaling pathway immobilized on a support, and areagent to perform, detect or quantify an immune reaction.
 14. Themethod of claim 8, wherein the method detects or diagnoses ahypopigmentation disorder and comprises the steps of: a) analysing theexpression of at least one protein of the WNT signaling pathway in abiological sample from a subject suspected of suffering of ahypopigmentation disorder, b) analysing the expression of the at leastone protein of the WNT signaling pathway in a biological sample from ahealthy subject, c) comparing the expressions of the at least oneprotein of the WNT signaling pathway as analysed in steps a) and b), anexpression of at least one protein of the WNT signaling pathway in thebiological sample from the subject suspected of suffering of apigmentation disorder lower than the expression of at least one proteinof the WNT signaling pathway in the biological sample from the healthysubject being an indicator of the presence of a hypopigmentationdisorder in the subject suspected of suffering of a hypopigmentationdisorder, thereby detecting or diagnosing the hypopigmentation disorder.15. A method for monitoring in vitro or ex vivo the course of ahypopigmentation disorder affecting a subject, wherein the methodcomprises a step of comparing the expression of at least one protein ofthe WNT signaling pathway in a first biological sample taken from asubject at t0 to the expression of the at least one protein of the WNTsignaling pathway in a second biological sample taken from said subjectat t1, a decrease of the expression of the at least one protein of theWNT signaling pathway in the sample taken at t1 being an indicator ofthe progression of the hypopigmentation disorder in said subject and anincrease of the expression of at least one protein of the WNT signalingpathway in the sample taken at t1 being an indicator of the regressionof the hypopigmentation disorder in said subject.
 16. A method formonitoring in vitro or ex vivo the efficacy of a drug or composition fortreating a hypopigmentation disorder, comprising comparing theexpression of at least one protein of the WNT signaling pathway in afirst biological sample from a subject identified as having one or moreof the symptoms of a hypopigmentation disorder before any treatment ofthe hypopigmentation disorder to the expression of the at least oneprotein of the WNT signaling pathway in a second biological sample ofthe same subject who has been exposed to a drug or composition fortreating a hypopigmentation disorder, an increase in the expression ofthe at least one protein of the WNT signaling pathway in the secondbiological sample being an indicator of efficacy of the drug orcomposition for treating the hypopigmentation disorder, and a decreasein the expression of the at least one protein of the WNT signalingpathway in the second biological sample being an indicator of inefficacyof the drug or composition for treating the hypopigmentation disorder.17. (canceled)
 18. An in vitro or ex vivo screening method of activatorsof a protein of the WNT signaling pathway, comprising the followingsteps of: contacting a biological sample exhibiting a hypopigmentationdisorder lesion, a biological sample healthy control sample, or amixture of said samples, with one or more drug candidates to be tested;detecting the expression and/or biological function of a protein of theWNT signaling pathway in the biological samples or mixture of thesamples comparing said expression and/or biological function with theexpression or biological function of the protein of the WNT signalingpathway in a sample which has not been contacted with the one or moredrug candidates; and selecting as activators of a protein of the WNTsignaling pathway drug candidates which increase the expression and/orbiological function of the protein of the WNT signaling pathway.
 19. Themethod of claim 13, wherein the reagent is an antibody.